Changes in mucin-type O-linked glycosylation of proteins are known to be linked to cancer. However, the complexity associated with mucin-type glycosylation and the limited tools available to study such glycoproteins has severely limited our understanding of their involvement in disease. The search for biomarkers is becoming a major focus in the field of mass spectrometry-based proteomics. However, changes in protein expression levels have been the main focus in this field. More recently, metabolic engineering and mass spectrometric techniques have been developed to study glycan structure. These techniques hold promise for glycobiologists studying changes to glycan structure related to biomarkers. Our preliminary studies have shown capture of metabolically incorporated O-linked azidosugars by Staudinger Ligation or "click chemistry" provides a method of detection for glycoproteins using mass spectrometry (MS). We propose a mass spectrometry-based method to detect biomarkers of cancer associated with changes to glycan structure of glycoproteins in cultured cells and a murine liver cancer model. Specific aims of the proposal are (1) Develop a method for identifying mucin-type glycosylation from complex lysates. (2) Develop a method to quantify changes in protein and glycosylation levels in complex lysates through stable isotope labeling and mass spectrometry. (3) Analyze changes in mucin-type glycosylation levels in a murine liver cancer model. Incorporating azidosugars into glycoproteins through metabolic engineering followed by a selective chemical reaction via "click chemistry" with a cleavable affinity tag will provide us with a method for the detection of glycoproteins using mass spectrometry that are not necessarily the most abundant proteins, but those that are produced at high flux. The use of a stable isotope labeled alkynyl tag with a unique signature will allow us to detect and quantify glycosylated and non-glycosylated peptides and compare differences in expression levels between healthy and diseased samples from cultured cells and a murine liver cancer model, the TRE-MYCxLAP-TTA mouse. The proposed project will contribute to our understanding of mucin-type O-linked glycosylation both from a basic science perspective as well as the discovery of cancer biomarkers. PUBLIC HEALTH RELEVANCE As sugars are found on proteins in nearly all mammalian cells, it is possible to find biomarkers of cancer in virtually any type of tissue. The discovery of reliable biomarkers for early detection of disease would undoubtedly improve the accuracy of diagnosis and long-term patient survival, and provide therapeutic targets for drug development.